The present invention relates to hybrid nucleic acid sequences, comprising at least the coding region of an unedited mitochondrial gene from higher plants and allowing the control of male fertility in plants containing the said sequences, to the transgenic plants having such sequences, as well as to a method for producing transgenic male-sterile plants and to a method for restoring male-fertile plants.
The control of male fertility in plants is one of the key problems for obtaining hybrids, and more particularly male-sterile lines which are of agronomic interest especially for controlling and improving seeds. Indeed, the large scale production of hybrid seeds with controlled characteristics is a real challenge since many crops have both male and female reproductive organs (stamens and pistils). This causes a high rate of self-pollination and makes difficult the control of crossings between lines for obtaining the desired hybrids.
In order to allow non-inbred crossings to be obtained which make it possible to produce hybrid seeds having advantageous properties, the inventors have developed new transgenic male-sterile plants capable of being restored and which facilitate the development of hybrid crops.
Cytoplasmic male sterility (MCS) is characterized by non-formation of the pollen after meiosis.
In alloplasmic systems, MCS is due to a nucleus-cytoplasm incompatibility which may occur at several levels: replication of DNA, transcription of genes, maturation of transcripts, translation or assembly of multiprotein complexes.
From the observations made on maize and petunia (Dewey R. E. et al., Cell, 1986, 44, 439; Young E. G. et al., Cell. 1987, 50, 41), comes the hypothesis that MCS is due to a deficiency in the mitochondrial bioenergetic machinery. Indeed, MCS manifests itself by a reduction in the ATP and NADP levels. At the cellular level, this deficiency is correlated with degeneration of the cells of the anther lawn, while having no effect on the development of the plant.
A number of methods have been proposed in the prior art for obtaining male-sterile plants.
There may be mentioned especially the backcrossings which lead to the substitution of the nuclear genome of a species by another genome and this, in the cytoplasmic environment of the first species (alloplasmy); this substitution may also appear spontaneously in field crops. MCS can also be obtained by protoplast fusion (Lonsdale D. M., Genetic Engineering, 1987, 6, 47).
In all these situations, the results are not reliable or reproducible; furthermore, in all cases, the manipulations are long, tedious and often difficult to control.
Male-sterile plants have also been obtained by transgenosis, with the aid of a gene encoding an RNAse, under the control of an anther-specific promoter (Mariani C. et al., Nature, 1990, 347, 737). This transgene, when expressed, has a toxic effect on the cell insofar as the endogenos RNAs are degraded, thereby causing cell death.
Another system, which also introduces a new artificial and destructive function, has been described by Worrall D. et al., (The Plant Cell, 1992, 4, 759-771) (callase system) and has the same disadvantages as the RNAse system.
Other methodologies have also been proposed forobtaining male-sterile plants; there may be mentioned especially the techniques which take advantage of the disruption of certain metabolic pathways (Van de Meer I. M. et al., The Plant Cell, 1992, 4, 253-262) (expression of a chalcone synthase antisense gene) or the techniques involving asymmetric somatic hybridization (Melchers C. et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 6832-6836) to bring into contact, as in alloplasmic male-sterile lines, the cytoplasm of a donor individual and the nucleus of a recipient partner. The latter two processes have the major disadvantage of being highly unpredictable as regards the desired objective, namely the obtaining of male-sterile plants which makes it possible to control reproduction in these plants.
The Applicant consequently set itself the objective of obtaining transgenic male-sterile plants in a controlled, reliable and reproducible manner which are capable of being used in agronomic programmes of seed improvement.
The subject of the present invention is transgenic plants having in their nuclei an expressible hybrid sequence (transgene) comprising at least one coding region of an unedited mitochondrial gene from higher plants and a sequence capable of transferring the protein expressed by the said coding region to the mitochondrion, which plants are characterized in that:
the coding regions of the unedited mitochondrial genes are chosen from among the genes encoding a protein of the ATP synthase complex which are chosen from among the wheat ATP9 gene fragment, of the following formula I:
or the ATP6 gene, or from among the genes encoding a protein of the respiratory chain which are chosen from among the genes for subunits 1 to 7 of NAD dehydrogenase, the gene for apocytochrome b and the genes for subunits I, II or III of cytochrome oxidase and
the sequence capable of transferring the said expressed protein to the mitochondrion is selected from the group consisting of the fragments encoding yeast tryptophanyl tRNA synthetase (SCHMITZ, U. K. et al., 1989, The Plant Cell, 1, 783-791), and the .beta. subunit of Nicotiana plumbaginifolia ATPase (BOUTRY et al., 1987, Nature, 328:340-342), and the maize ATP/ADP translocator (BATHGATE et al., 1989, Eur. J. Biochem., 183:303-310) or a 303 base pair EcoRI/KpnI fragment including codons 1 to 62 of subunit IV of yeast cytochrome oxidase (MAARSE et al., 1984, EMBO J., 3, 2831-2837),
which hybrid sequence is capable of modifying male fertility in plants having incorporated the said transgene while not modifying the other phenotypic characteristics of the said plants.